A genetic and molecular analysis of the process of DNA replication in yeast will allow the identification of the factors important for the regulation of eukaryotic DNA replication. Knowledge of this complex control mechanism will provide insight into the regulation of cell proliferation. Therefore, these studies will provide a basis for the understanding of oncogenic transformation, tissue regeneration and quiescient cell activation during a viral infection. Through a combination of procedures which include density-labelling of replicative intermediates, DNA restriction mapping and genomic Southern analysis the DNA sequences which comprise the origins of DNA replication will be identified. Genetic analysis of the DNA initiation process will focus on the effect of mutations in DNA replication proteins on the activation of the origins during the yeast S phase. The CDC7 gene product is a good candidate for a protein which acts as an activator molecule during initiation. The effects of different physiological treatments, for example the method of cell synchronization, on the pattern of origin activation will reveal the specific parameters which are important for the regulation. It is clear from prokaryotic studies that many DNA replication proteins act in concert via a multiprotein complex which assures a round of rapid, efficient and faithful DNA synthesis in each cell cycle. Evidence will be obtained for the existence of a yeast multiprotein complex by biochemical, immunologic and genetic methods. Biochemical assays can be used to identify DNA replication proteins in large molecular weight complexes isolated in vitro. The presence in the complex of other gene products for which an assay does not exist will be shown by constructing hybrid genes with the E. coli lacZ or trpE genes. Fusion proteins will be produced by expression of the hybrid genes in E. coli. Antibodies which are produced to the fusion proteins will be used as probes for identifying the protein in the complex. Molecular analysis of multiprotein complexes isolated from yeast cells which produce the fusion protein will be used to determine the DNA sequences important for participation in the complex. In vivo genetic analysis of extragenic suppressors of many DNA replication mutations will corroberate the protein relationships established in vitro, and also establish new protein interactions. Finally, a method for the selective enrichment of DNA replication mutants by use of chemotherapeutic drugs will be developed.